模拟在无帽依赖翻译中使用的 FGF-9 5'-UTR RNA 的结构和 DAP5 结合位点。

Modeling the structure and DAP5-binding site of the FGF-9 5'-UTR RNA utilized in cap-independent translation.

机构信息

PhD Program in Biochemistry, The Graduate Center, CUNY, New York, New York 10016, USA.

Department of Chemistry, Hunter College, CUNY, New York, New York 10065, USA.

出版信息

RNA. 2024 Aug 16;30(9):1184-1198. doi: 10.1261/rna.080013.124.

Abstract

Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5'-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.

摘要

帽非依赖性或真核起始因子(eIF)4E 非依赖性,真核生物的翻译起始需要支架蛋白 eIF4G 或其同源物,死亡相关蛋白 5(DAP5)。eIF4G 与 40S 核糖体亚基结合,将核糖体招募到 RNA 转录物上。一些 RNA 转录物,如成纤维细胞生长因子 9(FGF-9),包含直接与 DAP5 或 eIF4GI 结合的 5'非翻译区(5'UTR)。对于病毒 mRNA,eIF 募集通常利用 RNA 结构,如假结或茎环,并且 RNA 解旋酶 eIF4A 是 DAP5 或 4G 介导的翻译所必需的,这表明这些 5'UTR 是结构化的。然而,对于细胞 IRES 样翻译,尚未为 eIF 结合鉴定出共识 RNA 结构或序列。然而,FGF-9 5'UTR 中的 DAP5 结合位点是未知的。此外,DAP5 结合到其他不同的 5'UTR,其中一些需要未配对的、可及的 5'端来刺激帽非依赖性翻译。使用 SHAPE-seq,我们在体外模拟了 186nt FGF-9 5'-UTR RNA 的复杂二级结构。此外,DAP5 足迹、toeprinting 和 UV 交联实验鉴定了 DAP5-RNA 相互作用。FGF-9 5'-UTR 三级结构的建模将 DAP5 相互作用的核苷酸排列在预测结构的一个面上。我们提出,涉及三级折叠的 RNA 结构,而不是保守序列或二级结构,充当 DAP5 结合位点。DAP5 似乎与起始密码子附近的核苷酸接触。我们的发现为寻找帽非依赖性翻译增强子提供了新的视角。结构而非序列特异性的 eIF 结合位点可能成为有吸引力的化疗靶点或作为基于 mRNA 的治疗的剂量工具。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ca61/11331406/2a8df45b7c9c/1184f01.jpg

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